Abstract

Tregs hold great promise as a cellular therapy for multiple immunologically mediated diseases, given their ability to control immune responses. The success of such strategies depends on the expansion of healthy, suppressive Tregs ex vivo and in vivo following the transfer. In clinical studies, levels of transferred Tregs decline sharply in the blood within a few days of the transfer. Tregs have a high rate of apoptosis. Here, we describe a new mechanism of Treg self-inflicted damage. We show that granzymes A and -B (GrA and GrB), which are highly upregulated in human Tregs upon stimulation, leak out of cytotoxic granules to induce cleavage of cytoplasmic and nuclear substrates, precipitating apoptosis in target cells. GrA and GrB substrates were protected from cleavage by inhibiting granzyme activity in vitro. Additionally, we show — by using cytometry by time of flight (CYTOF) — an increase in GrB-expressing Tregs in the peripheral blood and renal allografts of transplant recipients undergoing rejection. These GrB-expressing Tregs showed an activated phenotype but were significantly more apoptotic than non–GrB expressing Tregs. This potentially novel finding improves our understanding of Treg survival and suggests that manipulating Gr expression or activity might be useful for designing more effective Treg therapies.

Figure 6

Western blots of cell lysates from the same biological sample (i.e., stimulated Tregs) were contemporaneously run on different gels, showing different GrB and GrA targets in the cytoplasm, mitochondria, and nucleus. For blots with lysates on continuous lanes, the conditions have been separated by a thin black line, while noncontiguous lanes on different blots have been separated by white space. The Tregs were cultured for 3 days in the presence or absence of the pan-granzyme inhibitor DCI, and the lysates were harvested at 0.5, 1, 2, and 3 days. β-Actin was used as the housekeeping protein. We observed that the cleavage of caspase 3, Bid, and Lamin A/C were inhibited in the presence of DCI, reflecting the physiological role of the Grs in this cleavage. These findings affirm the role of the Grs in cleaving caspase 3 and in causing apoptosis in stimulated Tregs, notably within 2 days of stimulation. Bid, BH3 interacting–domain death agonist; DCI, 3,4-dichloro-isocoumarin; h, hour; kDa, kilodalton; Parp1, poly(ADP-ribose) polymerase 1.